Gradients of cell signaling molecules and regulatory factors are pervasively used in a variety of patterning process in metazoan development. Examples include the Sonic Hedgehog gradient that specifies different neuronal cell types in the neural tube of vertebrate embryos, and the Dpp gradient that patterns the developing wing imaginal disk in Drosophila larvae. The Toll-Dorsal signaling pathway represents one of the most thoroughly characterized gradient systems in animal development. The proposed study represents a continuation of our efforts to determine how the Dorsal regulatory gradient produces multiple thresholds of gene expression and tissue differentiation in the early Drosophila embryo. The research plan includes 3 specific aims. First, we will examine the activities of Dorsal target genes that were identified in recent whole-genome microarray assays. Among the newly identified genes that will be tested is Eiger, the major TNF homolog in the Drosophila genome, and Mes4, which encodes a tissue-specific subunit of the CACAAT-box binding protein complex, NF-Y. Second, we will use a combination of bioinformatics methods and transgenic assays to determine how the Dorsal gradient regulates the expression of different target genes in a concentration-dependent manner. Particular efforts will focus on defining a "cis-regulatory code", whereby coordinately regulated target enhancers share a characteristic combination of factor binding sites, including binding sites for Dorsal and additional, mainly unknown regulatory factors. Third, we will determine whether immunity genes that are induced upon infection or injury share a similar organization of cis-regulatory elements. Particular efforts will focus on genes that are activated by the Dorsal-related immunity genes, Dif and Relish, and the GATA transcription factor, Serpent.